Detention of HPV L1 Capsid Protein and hTERC Gene in Screening of Cervical Cancer

Objective(s): To investigate the expression of human papilloma virus (HPV) L1 capsid protein, and human telomerase RNA component (hTERC) in cervical cancer and the role of detection of both genes in screening of cervical cancer. Materials and Methods: A total of 309 patients were recruited and cervical exfoliated cells were collected. Immunocytochemistry was employed to detect HPV L1 capsid protein, and fluorescent in situ hybridization (FISH) was performed to detect the hTERC. Results: The expression of HPV L1 capsid protein reduced with the increase of the histological grade of cervical cells and was negatively related to the grade of cervical lesions. However, the expression of hTERC increased with the increase of the histological grade and positively associated with the grade of cervical lesions. The proportion of patients with L1(-)/hTERC(+) was higher in patients with histological grade of CIN2 or higher than that in those with histological grade of CIN1. The L1(+)/hTERC(-) and L1(-)/hTERC(-) were negatively related to the grade of cervical lesions. L1(-)/hTERC(+) was positively associated with the grade of cervical lesions. The L1/hTERC ratio increased. The negative predictive value of both HPV L1 and hTERC was higher than that of HPV L1 or hTERC, but there was no marked difference in the screening efficacy of cervical cancer among HPV L1, hTERC and HPV L1+hTERC. Conclusion: HPV L1 capsid protein and hTERC gene may serve as markers for the early diagnosis and prediction of cervical lesions. The increase in L1/hTERC ratio reflects the progression of cervical lesions to a certain extent.


Introduction
Cervical cancer is the second leading common cause of malignancies in women (1)(2). World Health Organization (WHO) introduced cervical cancer as the first cancer which is entirely caused by infection (3). The progression of cervical precancerous lesion into cervical cancer is mainly attributed to the sustained infection of human papilloma virus (HPV). However, not all the HPV infection of cervix develops into cervical cancer and the majority of HPV infection is subclinical. Thus, to identify HPV infection, patients with high risk of cervical cancer, and then to reduce over-medication are the challenges in clinical practice (4)(5)(6)(7)(8). Studies found that HPV L1 capsid protein and human telomerase RNA component (hTERC) gene were the biomarkers of prognosis of HPV infection, but detection of both HPV L1 capsid protein and hTERC gene is seldom reported as a strategy for the prediction of cervical cancer (9)(10)(11)(12)(13)(14). In the present study, cervical exfoliated cells were collected from 309 patients and the expression of HPV L1 capsid protein and hTERC gene was determined. This study aimed at detecting the expression of HPV L1 capsid protein and hTERC gene in cervical exfoliated cells, and the role of detection of both HPV L1 capsid protein and hTERC gene in screening of cervical cancer.

Immunocytochemistry for HPV L1 Capsid Protein
CytoReact cell/tissue HPV L1 detection kit (Myprice Medical Technology Co., Ltd, USA) was used to detect HPV L1 capsid protein according to manufacturer's instructions. In brief, the smeared cells were fixed in 96% ethanol for 20 min and then dried. Subsequently, these cells underwent dehydration in 96%, 75% and 50% ethanol series and then in distilled water (2 min for each). Following washing with acid, pre-hybridization was done and inactivation of endogenous peroxidase was performed to remove the non-specific staining. Exogenous HPV L1 antibody was used to treat these cells followed by DNA hybridization and treatment with exogenous antibody again. Following hybridization, treatment with RNAase and peroxidase was performed. Staining was done with alkaline phosphatase and immunohistochemistry by AEC staining. Counterstaining and mounting were performed and sections were preserved at room temperature in dark. These sections were observed under a microscope. Sections with one nucleus stained red were regarded as positive for HPV L1. In addition, the negative and positive samples provided by the manufacture served as negative and positive controls, respectively and observed under a microscope. Sections with one nucleus stained red were regarded as positive for HPV L1.

Detection of hTERC gene with fluorescent in situ hybridization (FISH)
Probes (hTERC/CSP3 DNA) were provided by the Beijing Jinpujia Medical Technology Company. These probes can hybridize into 3q26.3, and red fluorescence (Tetramethylrhodamine) was presented. CSP3 served as a control probe, can hybridize into the centromere of chromosome 3 and presented green fluorescence. In brief, the cervical exfoliated cells were digested in collagenase B, treated with hypotonic solution and then fixed. Then cells were digested in pepsin. Following drying, cells were denaturated in formamide. The sections were hybridized with pre-denaturated probes overnight.
After washing, the sections were observed under a fluorescence microscope. Normal cells had single nucleus and presented one red and one green signal. Cells with abnormal hTERC amplification had single nucleus with more than two red signals and no less than two green signals. A total of hundred cells with complete signals were counted and was defined positive when six presented cells were increased in hTERC copies (this threshold was determined after reviewing normal cervical cells from 20 patients Detection of HPV L1 capsid protein and/or hTERC in diagnosis of CIN grade 2 or higher When diagnosis was done with findings in hTERC detection, the specificity, positive predictive value and accuracy were relatively high but the sensitivity was low (P<0.001). When diagnosis was done with findings in detection of HPV L1 capsid

Discussion
The incidence of cervical cancer is increasing gradually in the developing countries, and early examination and early diagnosis of cervical cancer and precancerous lesions play an important role in clinical practice (15)(16)(17)(18)(19). Increasing attention has been paid to the role of HPV L1 capsid protein in the early diagnosis of cervical cancer (20)(21)(22)(23). In the present study, the positive rate of HPV L1 capsid protein decreased with the increase in histological grade. In CIN1 patients, the HPV L1 capsid protein expression was higher than that in patients with CIN2/3 or SCC. These findings suggest that the expression of HPV L1 capsid protein is lost in CIN2/3 and SCC. HPV L1 is the major capsid protein of HPV (9,(24)(25)(26) and is expressed in early replication. When the HPV DNA is integrated into host DNA, the expression of HPV L1 capsid protein is lost in proliferative cells, and HPV L1 capsid protein is also a main target antigen inducing immune response following HPV infection (27)(28)(29)(30)(31)(32). Our results showed that HPV L1 expression was negatively related to the grade of cervical lesions, which indirectly confirmed that the deficiency of HPV L1 was attributed to the progression of cervical lesions. McMurry et al (33) and Hagensee et al (34) explained that the loss of HPV L1 expression in early transformation phase could lead to an ineffective stimulation of immune response. Thus, the loss of HPV L1 expression may compromise the cellular immunity inducing the progression of cervical lesions. Thus, HPV L1 can be used as a biomarker in early examination and prediction of cervical lesions. hTERC locates at 3q26 chromosome region and encodes hTR which is a template in the synthesis of telomeric repeat and a core component in maintenance of telomerase activity. In the progression of cervical cells from atypical dysplasia to cervical cancer, the amplification of hTERC is almost inevitable and has been a promising candidate gene of atypical canceration of cervical cells (35). Our findings indicated the hTERC was positively related to the grade of cervical lesions, and the hTERC level increases with the increase in histological grade of cervical lesions. This also suggests that hTERC is a tumor marker in the malignant transformation of cervical intraepithelial neoplasia cells. Thus, hTERC can be used as a biomarker for the early diagnosis and the prediction of cervical lesions.
In the present study, histological findings served as gold standard and CIN grade two or higher served as positive and CIN grade one or lower as negative. When the diagnosis was performed with findings in hTERC detection alone, the specificity, positive predictive value and accuracy were relatively high, and detection of both HPV L1 capsid protein and hTERC had relatively high negative predictive value. Thus, the diagnostic value of detection of HPV L1 capsid protein and/or hTERC was compared with AUC of ROC. Results showed that the detection of HPV L1 capsid protein and/or hTERC could be used in the diagnosis of CIN gradetwo or higher, but the detection of both HPV L1 capsid protein and hTERC did not further increase the accuracy. This may be attributed to the negative for HPV L1 protein which reflects two statuses of viral DNA: viral DNA was integrated into host DNA resulting in malignant transformation; latent infection or absence of viral replication. This may also explain why the specificity and accuracy of HPV L1 capsid protein detection were lower than those of hTERC detection alone.
In addition, our results also revealed that L1 (-)/hTERC(+) was positively related closely to the grade of cervical lesions (rs=0.612,P<0.001). In CIN1 patients, the proportion of L1(-)/hTERC(+) patients was lower than that in patients with CIN2/3 (42.9%) or SCC (100%), and this was the unique form in SCC patients. This indicates the loss of HPV L1 expression, and hTERC amplification plays an important role in the progression of cervical lesions. L1(-)/hTERC(+) also demonstrates that the HPV DNA has been integrated into host genome, and loss of HPV L1 expression and hTERC amplification may predict a high risk for cervical cancer. L1(+)/hTERC(+) was only found in CIN patients but not in SCC patients and those with normal cervical cells or chronic cervicitis. L1(+)/hTERC(+) means the presence of hTERC amplification, replication of HBP DNA, synthesis of HPV L1 and assembly of virosome. This form predicts high infectivity but can not be used to differentiate low grade from high grade.

Conclusion
The expression of HPV L1 capsid protein reduced with the increase of the histological grade of cervical cells and was negatively related to the grade of cervical lesions. However, the expression of hTERC increased with the increase of the histological grade and positively associated with the grade of cervical lesions. HPV L1 capsid protein and hTERC gene may serve as markers for the early diagnosis and prediction of cervical lesions. The increase in L1/hTERC ratio reflects the progression of cervical lesions to a certain extent.